Objective To characterize the hydrolysis product and the substrate binding in the catalytic cavity of alpha-agarase AgaD. Results The time course curve showed that AgaD degraded agarose by the endo-type cleavage. AgaD did not degrade agarobiose (A2) and agarotetraose (A4). The minimum-length substrate was agarohexaose (A6), which was cleaved into A2 and A4. Agarooctaose (A8) was cleaved into two molecules of A4. Consistently, TLC and NMR data identified agarotetraose (A4) as the main hydrolysate when agarose was degraded by AgaD. Conclusion This study confirms AgaD is an endo-type alpha-agarase and A4 as the main hydrolysis product of AgaD, which suggests the catalytic cavity of AgaD accommodates eight sugar units spanning from - 4 to + 4.